Modulation of estrogen-responsive finger protein expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of estrogen-responsive finger protein. The compositions comprise oligonucleotides, targeted to nucleic acid encoding estrogen-responsive finger protein. Methods of using these compounds for modulation of estrogen-responsive finger protein expression and for diagnosis and treatment of disease associated with expression of estrogen-responsive finger protein are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of estrogen-responsive finger protein. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding estrogen-responsive finger protein. Such compounds are shown herein to modulate the expression of estrogen-responsive finger protein.

BACKGROUND OF THE INVENTION

[0002] The degradation of proteins which are no longer needed in a cell is an important regulatory mechanism for cellular processes such as cell cycle progression, DNA repair, stress response, organelle biogenesis, apoptosis, inflammation, transcription, and signal transduction. The 26S proteasome is responsible for most of the non-lysosomal degradation of intracellular proteins. Many proteins that are targeted for destruction by the 26S proteasome bear a polyubiquitin chain on lysine residues which acts a signal for protein destruction. Ubiquitin is a 76-amino acid globular protein that is highly conserved in eukaryotes and the defective regulation of the ubiquitin pathway is manifested in diseases ranging from developmental abnormalities, autoimmunity, neurodegenerative diseases, and cancer (Weissman, Nat Rev Mol Cell Biol, 2001, 2, 169-178).

[0003] The covalent ligation of ubiquitin to target proteins involves three separate steps. First, the carboxy terminus of ubiquitin is activated via thiol ester bond formation in an ATP-dependent manner by a ubiquitin-activating enzyme from the E1 family. Second, the activated ubiquitin is transferred via this thiol ester bond to a ubiquitin carrier protein from the E2 family. Third, a ubiquitin protein ligase from the E3 family catalyses the transfer of ubiquitin from the E2 enzyme to the target protein. Each of these families, E1, E2, and E3, contains multiple enzymes which permits a great degree of specific and sensitive substrate recognition to maintain the integrity of this ubiquitous process (Weissman, Nat Rev Mol Cell Biol, 2001, 2, 169-178).

[0004] One of these E3 ligases, estrogen-responsive finger protein, may be a key protein controlling the switch from estrogen-dependent to estrogen-independent growth in tumors. The gene encoding estrogen-responsive finger protein (also called Efp, estrogen-responsive zinc-finger protein, zinc finger protein 147, ZNF147, and TRIM25) was cloned in 1993 (Inoue et al., Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 11117-11121) and the genomic structure was further described in 2000 (Ikeda et al., FEBS Lett., 2000, 472, 9-13). The protein contains a consensus estrogen-responsive element in the 3′ untranslated region that can act as a downstream estrogen-dependent enhancer, and a RING finger motif which is present in a family of apparent DNA-binding proteins, and this led to the suggestion that estrogen-responsive finger protein is an estrogen-responsive transcription factor (Inoue et al., Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 11117-11121). Cloning of the 5′ flanking region revealed the promoter structure lacks a TATA box, but contains an E-box and a GC-rich regulatory region (Ikeda et al., Biochem. Biophys. Res. Commun., 1997, 236, 765-771). The gene has been mapped to chromosomal location 17q21.3-q22 (Inoue et al., Genomics, 1995, 25, 581-583), a region amplified in ovarian cancer cell lines (Watanabe et al., Gynecol. Oncol., 2001, 81, 172-177).

[0005] The proposed mechanism for the involvement of estrogen-responsive finger protein in the switch from estrogen-dependent to estrogen-independent growth in breast tumors results from its role as an E3 ubiquitin ligase. Estrogen-responsive finger protein targets proteolysis of 14-3-3sigma, a negative cell cycle regulator which causes G2 arrest (Urano et al., Nature, 2002, 417, 871-875). Estrogen-responsive finger protein has also been found to be upregulated in breast cancer (Thomson et al., Int. J. Cancer, 2001, 91, 152-158), and induced in gastrointestinal carcinoma cells following treatment with bile acids (Jung et al., Carcinogenesis, 1998, 19, 1901-1906). In breast cancer cell lines, estrogen-responsive finger protein promoter activity has been found to be enhanced through the estrogen-responsive element dependent on estrogen and estrogen receptor (Ikeda et al., FEBS Lett., 2000, 472, 9-13).

[0006] Mice carrying a loss-of-function mutation in estrogen-responsive finger protein indicated that it is essential for the normal estrogen-induced cell proliferation and uterine swelling as estrogen-responsive finger protein is one of the direct targets of estrogen receptor alpha (Orimo et al., Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 12027-12032).

[0007] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of estrogen-responsive finger protein and to date, investigative studies aimed at modulating estrogen-responsive finger protein function have involved the use of an antisense strategy. Loss of estrogen-responsive finger protein function in mouse embryonic fibroblasts by treatment with a phosphorothioated antisense oligonucleotide targeted to the mouse mRNA resulted in an accumulation of 14-3-3sigma and reduced tumor growth (Urano et al., Nature, 2002, 417, 871-875).

[0008] Consequently, there remains a long felt need for additional agents capable of effectively inhibiting estrogen-responsive finger protein function.

[0009] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of estrogen-responsive finger protein expression.

[0010] The present invention provides compositions and methods for modulating estrogen-responsive finger protein expression.

SUMMARY OF THE INVENTION

[0011] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding estrogen-responsive finger protein, and which modulate the expression of estrogen-responsive finger protein. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of estrogen-responsive finger protein and methods of modulating the expression of estrogen-responsive finger protein in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of estrogen-responsive finger protein are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0012] A. Overview of the Invention

[0013] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding estrogen-responsive finger protein. This is accomplished by providing oligonucleotides which specifically hybridize with one,or more nucleic acid molecules encoding estrogen-responsive finger protein. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding estrogen-responsive finger protein” have been used for convenience to encompass DNA encoding estrogen-responsive finger protein, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0014] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of estrogen-responsive finger protein. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0015] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0016] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0017] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0018] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0019] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0020] B. Compounds of the Invention

[0021] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0022] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0023] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0024] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0025] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0026] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0027] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0028] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0029] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0030] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0031] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0032] C. Targets of the Invention

[0033] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes estrogen-responsive finger protein.

[0034] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0035] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding estrogen-responsive finger protein, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0036] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0037] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0038] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0039] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0040] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0041] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0042] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0043] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0044] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0045] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0046] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0047] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0048] D. Screening and Target Validation

[0049] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of estrogen-responsive finger protein. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding estrogen-responsive finger protein and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding estrogen-responsive finger protein with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding estrogen-responsive finger protein. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding estrogen-responsive finger protein, the modulator may then be employed in further investigative studies of the function of estrogen-responsive finger protein, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0050] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0051] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0052] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between estrogen-responsive finger protein and a disease state, phenotype, or condition. These methods include detecting or modulating estrogen-responsive finger protein comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of estrogen-responsive finger protein and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0053] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0054] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0055] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0056] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0057] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0058] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding estrogen-responsive finger protein. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective estrogen-responsive finger protein inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding estrogen-responsive finger protein and in the amplification of said nucleic acid molecules for detection or for use in further studies of estrogen-responsive finger protein. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding estrogen-responsive finger protein can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of estrogen-responsive finger protein in a sample may also be prepared.

[0059] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0060] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of estrogen-responsive finger protein is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a estrogen-responsive finger protein inhibitor. The estrogen-responsive finger protein inhibitors of the present invention effectively inhibit the activity of the estrogen-responsive finger protein protein or inhibit the expression of the estrogen-responsive finger protein protein. In one embodiment, the activity or expression of estrogen-responsive finger protein in an animal is inhibited by about 10%. Preferably, the activity or expression of estrogen-responsive finger protein in an animal is inhibited by about 30%. More preferably, the activity or expression of estrogen-responsive finger protein in an animal is inhibited by 50% or more.

[0061] For example, the reduction of the expression of estrogen-responsive finger protein may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding estrogen-responsive finger protein protein and/or the estrogen-responsive finger protein protein itself.

[0062] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0063] F. Modifications

[0064] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0065] Modified Internucleoside Linkages (Backbones)

[0066] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0067] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 31′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0068] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0069] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholinowlinkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0070] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0071] Modified Sugar and Internucleoside Linkages-Mimetics

[0072] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0073] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂— N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0074] Modified Sugars

[0075] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0076] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0077] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAS) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0078] Natural and Modified Nucleobases

[0079] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0080] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0081] Conjugates

[0082] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0083] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0084] Chimeric Compounds

[0085] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0086] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0087] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0088] G. Formulations

[0089] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0090] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0091] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0092] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0093] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0094] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0095] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0096] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0097] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0098] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0099] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0100] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0102] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0103] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0104] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0105] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0106] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0107] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0108] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0109] H. Dosing

[0110] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0111] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0112] Synthesis of Nucleoside Phosphoramidites

[0113] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl, dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy- N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,41-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N ⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylamino-oxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine , 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5,-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0114] Oligonucleotide and Oligonucleoside Synthesis

[0115] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0116] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0117] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphate linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0118] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0119] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0120] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0121] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0122] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0123] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0124] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0125] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0126] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0127] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0128] RNA Synthesis

[0129] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0130] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0131] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0132] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0133] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0134] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedron Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0135] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5×annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0136] Synthesis of Chimeric Oligonucleotides

[0137] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0138] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0139] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0140] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0141] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0142] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0143] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxy phosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0144] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0145] Design and Screening of Duplexed Antisense Compounds Targeting Estrogen-responsive Finger Protein

[0146] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target estrogen-responsive finger protein. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0147] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0148] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15uL of a 5×solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0149] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate estrogen-responsive finger protein expression.

[0150] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0151] Oligonucleotide Isolation

[0152] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32 +/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0153] Oligonucleotide Synthesis—96 Well Plate Format

[0154] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0155] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0156] Oligonucleotide Analysis—96-Well Plate Format

[0157] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0158] Cell Culture and Oligonucleotide Treatment

[0159] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0160] T-24 cells:

[0161] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0162] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0163] A549 cells:

[0164] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0165] NHDF cells:

[0166] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0167] HEK cells:

[0168] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0169] Treatment with Antisense Compounds:

[0170] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0171] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0172] Analysis of Oligonucleotide Inhibition of Estrogen-responsive Finger Protein Expression

[0173] Antisense modulation of estrogen-responsive finger protein expression can be assayed in a variety of ways known in the art. For example, estrogen-responsive finger protein mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0174] Protein levels of estrogen-responsive finger protein can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to estrogen-responsive finger protein can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0175] Design of Phenotypic Assays and In Vivo Studies for the Use of Estrogen-responsive Finger Protein Inhibitors

[0176] Phenotypic Assays

[0177] Once estrogen-responsive finger protein inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition.

[0178] Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of estrogen-responsive finger protein in health and disease.

[0179] Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0180] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with estrogen-responsive finger protein inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0181] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0182] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the estrogen-responsive finger protein inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0183] In Vivo Studies

[0184] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0185] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or estrogen-responsive finger protein inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a estrogen-responsive finger protein inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0186] Volunteers receive either the estrogen-responsive finger protein inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding estrogen-responsive finger protein or estrogen-responsive finger protein protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0187] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0188] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and estrogen-responsive finger protein inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the estrogen-responsive finger protein inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0189] RNA Isolation

[0190] Poly(A)+ mRNA Isolation

[0191] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0192] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0193] Total RNA Isolation

[0194] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0195] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0196] Real-time Quantitative PCR Analysis of Estrogen-responsive Finger Protein mRNA Levels

[0197] Quantitation of estrogen-responsive finger protein mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0198] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0199] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0200] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0201] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0202] Probes and primers to human estrogen-responsive finger protein were designed to hybridize to a human estrogen-responsive finger protein sequence, using published sequence information (nucleotides 5642706 to 5667000 of the sequence with GenBank accession number NT_(—)010783.9, incorporated herein as SEQ ID NO: 4). For human estrogen-responsive finger protein the PCR primers were: forward primer: GGCGTGCTTCTCAACTGTGA (SEQ ID NO: 5) reverse primer: TGGACCTTGTCGGCAACA (SEQ ID NO: 6) and the PCR probe was: FAM-CACGGCTTTGTCATCTTCTTCG-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0203] Northern Blot Analysis of Estrogen-responsive Finger Protein mRNA Levels

[0204] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™−N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0205] To detect human estrogen-responsive finger protein, a human estrogen-responsive finger protein specific probe was prepared by PCR using the forward primer GGCGTGCTTCTCAACTGTGA (SEQ ID NO: 5) and the reverse primer TGGACCTTGTCGGCAACA (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0206] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0207] Antisense Inhibition of Human Estrogen-responsive Finger Protein Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0208] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human estrogen-responsive finger protein RNA, using published sequences (nucleotides 5642706 to 5667000 of the sequence with GenBank accession number NT_(—)010783.9, incorporated herein as SEQ ID NO: 4, GenBank accession number NM_(—)005082.1, incorporated herein as SEQ ID NO: 11, and GenBank accession number BM552557.1, incorporated herein as SEQ ID NO: 13). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcitidines. The compounds were analyzed for their effect on human estrogen-responsive finger protein mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in where A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human estrogen-responsive finger protein mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB ID NO NO 285764 5′UTR 4 422 acacaactgctgcacccgcg 65 15 1 285765 3′UTR 11 2269 gatctgaaggctatggattt 13 16 1 285766 intron 4 1739 gtcaggctaagttcatttct 59 17 1 285767 intron 4 4551 tgggaaggaaaatcaccacc 80 18 1 285768 exon: 4 10190 ggcgacctacctccagaaac 23 19 1 intron junction 285769 intron 4 17170 tgccaccttccacaacactg 67 20 1 285770 exon: 4 18925 gataacttaccctccaagcc 35 21 1 intron junction 285771 intron 4 21798 attcatcagctcatctatcc 40 22 1 285772 5′UTR 4 425 gggacacaactgctgcaccc 63 23 1 285773 Start 4 456 gcacagctctgccatggcgc 58 24 1 Codon 285774 exon 4 478 acgacagctcctcggccagg 55 25 1 285775 exon 4 513 gaccggctccttgaagggct 67 26 1 285776 exon 4 539 cagaagttgtggccgcacgg 62 27 1 285777 exon 4 567 tgcccacgtctcattcaggc 71 28 1 285778 exon 4 669 gaactgctccaccacgttgc 76 29 1 285779 exon 4 674 tgcaggaactgctccaccac 63 30 1 285780 exon 4 679 cggcctgcaggaactgctcc 67 31 1 285781 exon 4 768 gtggtcgcaggccacctggg 66 32 1 285782 exon 4 807 gcacaccaagcacgtcttca 69 33 1 285783 exon 4 824 tgacagaaggaggccatgca 58 34 1 285784 exon 4 879 ctgcagcgggtggtcctgga 77 35 1 285785 exon 4 979 ggcagatgtggcagatgcac 68 36 1 285786 exon 4 1000 agcaggtcttatgctccacc 73 37 1 285787 exon 4 1005 gggagagcaggtcttatgct 35 38 1 285788 exon 4 5901 gtacatgacagttagtttgt 69 39 1 285789 Coding 11 880 ttgcagtcatccgcacatcc 70 40 1 285790 intron: 4 9965 cctttctgtttgcagtcatc 74 41 1 exon junction 285791 exon 4 10012 gtccaagagagccttcattt 65 42 1 285792 exon 4 10032 gtcgaggtggtctctgaggc 64 43 1 285793 exon 4 10068 ctgttgaccctcttctcctc 58 44 1 285794 exon 4 10094 gaatctgataaatggtgtca 65 45 1 285795 exon 4 10113 tcactcttcttcttgaggag 55 46 1 285796 exon 4 10127 tcaaggtctggatctcactc 64 47 1 285797 exon 4 10158 ctcttggtcaggctctgttc 86 48 1 285798 Coding 11 1161 ttgatgctttctccagaaac 67 49 1 285799 exon 4 12884 ttgagattcctcgcagtttt 66 50 1 285800 exon 4 12946 ctggtggatgccttttatca 76 51 1 285801 exon 4 12951 gtgctctggtggatgccttt 74 52 1 285802 exon 4 12959 ggtctatggtgctctggtgg 58 53 1 285803 exon 4 15281 acgctgggtcatgctctcca 86 54 1 285804 exon 4 15308 tcacagggcgtgtggatttg 72 55 1 285805 exon 4 18902 ttgttttaaatccactaact 43 56 1 285806 exon 4 19049 accttggccttgagagatgt 66 57 1 285807 exon 4 19091 tccaggagctcaggtctgga 76 58 1 285808 Coding 11 1570 ttaatgtaatactccaggag 25 59 1 285809 exon 4 22290 cagccacagaagctactgta 70 60 1 285810 exon 4 22303 ttctgaggcatctcagccac 40 61 1 285811 exon 4 22318 ggatgcggccggtagttctg 47 62 1 285812 exon 4 22340 agagcagtatgtgaacctct 78 63 1 285813 exon 4 22345 acctgagagcagtatgtgaa 77 64 1 285814 exon 4 22393 tccacctcccagtagtggat 74 65 1 285815 exon 4 22398 gcagctccacctcccagtag 85 66 1 285816 exon 4 22405 ttcttctgcagctccacctc 54 67 1 285817 exon 4 22410 agttgttcttctgcagctcc 77 68 1 285818 exon 4 22415 acagaagttgttcttctgca 68 69 1 285819 exon 4 22429 cagatgcctaccccacagaa 74 70 1 285820 exon 4 22475 gcggccgagcctgctttctg 62 71 1 285821 exon 4 22513 ttggtgttgaaccactccac 76 72 1 285822 exon 4 22550 cagggttttctccacgttat 80 73 1 285823 exon 4 22603 aagccgtggtcacagttgag 83 74 1 285824 exon 4 22615 aagaagatgacaaagccgtg 68 75 1 285825 exon 4 22663 aagtccaccctgaacttata 55 76 1 285826 exon 4 22695 atacccagaaagccgggtac 59 77 1 285827 exon 4 22714 agtgtggcaccagcagaaaa 66 78 1 285828 exon 4 22720 atggagagtgtggcaccagc 82 79 1 285829 Stop 4 22747 ctacagcctgcctacttggg 61 80 1 Codon 285830 3′UTR 4 22818 tccaaggagagttctgcctg 80 81 1 285831 3′UTR 4 22906 tcacccctttcctggctaaa 74 82 1 285832 3′UTR 4 23071 tatggattttctctaagagg 69 83 1 285833 3′UTR 4 23109 ttcagatccaagtggcccaa 62 84 1 285834 intron:e 4 23142 ctgaaggctaccccagagag 42 85 1 xon junction 285835 intron:e 4 23147 aagatctgaaggctacccca 50 86 1 xon junction 285836 3′UTR 4 23227 cgggtaatggagtttatgta 75 87 1 285837 exon 4 23283 tgctgtgggtgtttcctcaa 82 88 1 285838 exon 4 23462 ctgaatcacaaatccaacac 50 89 1 285839 genomic 13 729 accaggcttctgtgatccag 52 90 1 285840 intron 4 18779 cccttccacactgattaaga 66 91 1 285841 intron 4 18795 cgggatggcctctactccct 51 92 1

[0209] As shown in Table 1, SEQ ID NOs 15, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 and 92 demonstrated at least 35% inhibition of human estrogen-responsive finger protein expression in this assay and are therefore preferred. More preferred are SEQ ID NOs 48, 54 and 66. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target segments identified in estrogen-responsive finger protein. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 201822 4 422 cgcgggtgcagcagttgtgt 15 H. sapiens 93 201824 4 1739 agaaatgaacttagcctgac 17 H. sapiens 94 201825 4 4551 ggtggtgattttccttccca 18 H. sapiens 95 201827 4 17170 cagtgttgtggaaggtggca 20 H. sapiens 96 201828 4 18925 ggcttggagggtaagttatc 21 H. sapiens 97 201829 4 21798 ggatagatgagctgatgaat 22 H. sapiens 98 201830 4 425 gggtgcagcagttgtgtccc 23 H. sapiens 99 201831 4 456 gcgccatggcagagctgtgc 24 H. sapiens 100 201832 4 478 cctggccgaggagctgtcgt 25 H. sapiens 101 201833 4 513 agcccttcaaggagccggtc 26 H. sapiens 102 201834 4 539 ccgtgcggccacaacttctg 27 H. sapiens 103 201835 4 567 gcctgaatgagacgtgggca 28 H. sapiens 104 201836 4 669 gcaacgtggtggagcagttc 29 H. sapiens 105 201837 4 674 gtggtggagcagttcctgca 30 H. sapiens 106 201838 4 679 ggagcagttcctgcaggccg 31 H. sapiens 107 201839 4 768 cccaggtggcctgcgaccac 32 H. sapiens 108 201840 4 807 tgaagacgtgcttggtgtgc 33 H. sapiens 109 201841 4 824 tgcatggcctccttctgtca 34 H. sapiens 110 201842 4 879 tccaggaccacccgctgcag 35 H. sapiens 111 201843 4 979 gtgcatctgccacatctgcc 36 H. sapiens 112 201844 4 1000 ggtggagcataagacctgct 37 H. sapiens 113 201845 4 1005 agcataagacctgctctccc 38 H. sapiens 114 201846 4 5901 acaaactaactgtcatgtac 39 H. sapiens 115 201847 11 880 ggatgtgcggatgactgcaa 40 H. sapiens 116 291848 4 9965 gatgactgcaaacagaaagg 41 H. sapiens 117 201849 4 10012 aaatgaaggctctcttggac 42 H. sapiens 118 201850 4 10032 gcctcagagaccacctcgac 43 H. sapiens 119 201851 4 10068 gaggagaagagggtcaacag 44 H. sapiens 120 201852 4 10094 tgacaccatttatcagattc 45 H. sapiens 121 201853 4 10113 ctcctcaagaagaagagtga 46 H. sapiens 122 201854 4 10127 gagtgagatccagaccttga 47 H. sapiens 123 201855 4 10158 gaacagagcctgaccaagag 48 H. sapiens 124 201856 11 1161 gtttctggagaaagcatcaa 49 H. sapiens 125 201857 4 12884 aaaactgcgaggaatctcaa 50 H. sapiens 126 201858 4 12946 tgataaaaggcatccaccag 51 H. sapiens 127 201859 4 12951 aaaggcatccaccagagcac 52 H. sapiens 128 201860 4 12959 ccaccagagcaccatagacc 53 H. sapiens 129 201861 4 15281 tggagagcatgacccagcgt 54 H. sapiens 130 201862 4 15308 caaatccacacgccctgtga 55 H. sapiens 131 201863 4 18902 agttagtggatttaaaacaa 56 H. sapiens 132 201864 4 19049 acatctctcaaggccaaggt 57 H. sapiens 133 201865 4 19091 tccagacctgagctcctgga 58 H. sapiens 134 201867 4 22290 tacagtagcttctgtggctg 60 H. sapiens 135 201868 4 22303 gtggctgagatgcctcagaa 61 H. sapiens 136 201869 4 22318 cagaactaccggccgcatcc 62 H. sapiens 137 201870 4 22340 agaggttcacatactgctct 63 H. sapiens 138 201871 4 22345 ttcacatactgctctcaggt 64 H. sapiens 139 201872 4 22393 atccactactgggaggtgga 65 H. sapiens 140 201873 4 22398 ctactgggaggtggagctgc 66 H. sapiens 141 201874 4 22405 gaggtggagctgcagaagaa 67 H. sapiens 142 201875 4 22410 ggagctgcaqaagaacaact 68 H. sapiens 143 201876 4 22415 tgcagaagaacaacttctgt 69 H. sapiens 144 201877 4 22429 ttctgtggggtaggcatctg 70 H. sapiens 145 201878 4 22475 cagaaagcaggctcggccgc 71 H. sapiens 146 201879 4 22513 gtggagtggttcaacaccaa 72 H. sapiens 147 201880 4 22550 ataacgtggagaaaaccctg 73 H. sapiens 148 201881 4 22603 ctcaactgtgaccacggctt 74 H. sapiens 149 201882 4 22615 cacggctttgtcatcttctt 75 H. sapiens 150 201883 4 22663 tataagttcagggtggactt 76 H. sapiens 151 201884 4 22695 gtacccggctttctgggtat 77 H. sapiens 152 201885 4 22714 ttttctgctggtgccacact 78 H. sapiens 153 201886 4 22720 gctggtgccacactctccat 79 H. sapiens 154 201887 4 22747 cccaagtaggcaggctgtag 80 H. sapiens 155 201888 4 22818 caggcagaactctccttgga 81 H. sapiens 156 201889 4 22906 tttagccaggaaaggggtga 82 H. sapiens 157 201890 4 23071 cctcttagagaaaatccata 83 H. sapiens 158 201891 4 23109 ttgggccacttggatctgaa 84 H. sapiens 159 201892 4 23142 ctctctggggtagccttcag 85 H. sapiens 160 201893 4 23147 tggggtagccttcagatctt 86 H. sapiens 161 201894 4 23227 tacataaactccattacccg 87 H. sapiens 162 201895 4 23283 ttgaggaaacacccacagca 88 H. sapiens 163 201896 4 23462 gtgttggatttgtgattcag 89 H. sapiens 164 201897 13 729 ctggatcacagaagcctggt 90 H. sapiens 165 201898 4 18779 tcttaatcagtgtggaaggg 91 H. sapiens 166 201899 4 18795 agggagtagaggccatcccg 92 H. sapiens 167

[0210] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of estrogen-responsive finger protein.

[0211] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0212] Western Blot Analysis of Estrogen-responsive Finger Protein Protein Levels

[0213] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to estrogen-responsive finger-protein is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 168 1 20 DNA Artificial Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Antisense Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 24295 DNA Homo sapiens 4 tggtctcaga tgagtgctgg gaaggagggg acttgctccg agcgcaagtt tgtgcggaag 60 cgcggctgga cctgggctct gaatccgggg gtccggggtt ctgcacccag gcgtcagctt 120 cctcatccgc agagtggccc ccagaagcct ccgggtggtc gcgaggatgc tctaaatccc 180 gggggctaag gccgagcccg gcgtcccgcg cccagcccgc gggagctctt ggggatcgga 240 gcgcggccga ccttcgccag ctcatgggcg actgggactg cggtgcccgc aggctcccgg 300 gagtacctcg cgaggcccgc ccagtcctcg ccgcccgccc ccccgcaccc cacgtgactt 360 cctgacggct tcagggactg ctcctctcga gctaggtttc gtttcctcgg cggcctcgga 420 gcgcgggtgc agcagttgtg tcccgacccc tgggagcgcc atggcagagc tgtgccccct 480 ggccgaggag ctgtcgtgct ccatctgcct ggagcccttc aaggagccgg tcaccactcc 540 gtgcggccac aacttctgcg ggtcgtgcct gaatgagacg tgggcagtcc agggctcgcc 600 atacctgtgc ccgcagtgcc gcgccgtcta ccaggcgcga ccgcagctgc acaagaacac 660 ggtgctgtgc aacgtggtgg agcagttcct gcaggccgac ctggcccggg agccacccgc 720 cgacgtctgg acgccgcccg cccgcgcctc tgcacccagc ccgaatgccc aggtggcctg 780 cgaccactgc ctgaaggagg ccgccgtgaa gacgtgcttg gtgtgcatgg cctccttctg 840 tcaggagcac ctgcagccgc acttcgacag ccccgccttc caggaccacc cgctgcagcc 900 gcccgttcgc gacctgttgc gccgcaaatg ttcccagcac aatcggctgc gggaattttt 960 ctgccccgag cacagcgagt gcatctgcca catctgcctg gtggagcata agacctgctc 1020 tcccgcgtcc ctgagccagg ccagcgccga cctggaggta gggaacggcc tgctgggtgc 1080 agagggcagc ctggcctgat gggtggtgca gaggggccac cgggggcctc tctgccacac 1140 tctggacgcc ctggggagat gggcctgaat tcgaggccgg ggcatctcca atgtcagagt 1200 agaaagggga tgggagatta tatagtccct tcccatcatc catgaagaca ggttaggttg 1260 cttgccataa tcctgtcatt gtctgatggt gtttattttt atttttttga gacagggtct 1320 cgtccctctc acccaagctg gagtgcagtg gcggaatcac agctcactgc agcctcgact 1380 tcccgggctt aagggatcct cctacctcag cctcaagagt agctgggacc acagcctccc 1440 aagtagctgg gaccacaggc tcgctaattt tttgtagaga tgaggtttcg ccatgttgcc 1500 caggctggtc tcgaattcct gagctcaagc gatctgccca cctcgacctc ctaaagtgct 1560 aggaggtctt taaaggcgtg caccactgcg ccccaccatc tgatggtgtt tagtagaggg 1620 ttttgaatgt caactatgaa cgttggaggt tacctctgtt ccctcacgaa gtcctggtgg 1680 cacccaggag gtggatatga tttagtttca ttttactgtt gaggatactg aggtccagag 1740 aaatgaactt agcctgacct aatgcagtaa atagccacgc aggcattgtg acccgggttg 1800 gttcgatttc atagtttgca tttcttccca tggaccagcc tcgtatccag gtccgtagag 1860 tccggagcac caaacagtcc aggaagcttg aggtcatgta agcaaaagcc tttggtgggc 1920 cggagagttc ctgtgatcag gaagggttgt tattagaggc ctaagaggga gggtgggcag 1980 cgtcccagta atcactggat cagagccaca gggagagcca acactcttgc accatagtgt 2040 cctccgtcct ggccgtgtga ctttgagcat ggggtttggc ccctggacct gggtcctcac 2100 cagtatagtg aggctgtggg actaggtgga ttcctaaggg cctttcaggt ctgcaatgtt 2160 tttaggtggt ttggggatca taccttcccc ttgtggcctc aaagcagagg ctgaaatcaa 2220 accacctttt gttacatcag cctaagatcc aaagcatggc ctgatttcat ttcattctca 2280 cagcaccttc agggagaaca agacccgtct tcgtgttgtc agaggagaca gtagcctgag 2340 caaggtcaga gttgggggtc tgcatccagc tttgtgtagc atataaactt ggcttttttt 2400 ttcttttctt ttagagacag ggtcttgcta tgttgcctag gatgttcttg aacttccggc 2460 ctcaagggat cctcccatct cagcctcctg agtagctggg actacaggtg tgccaccgtg 2520 cctggctcta aacttggctt ttttttttct tcttttagag acaggatctt actctgtcac 2580 ccaggctgga gtacagtggg gtgatcatag ctcactgtaa tctcaaactc ctgggctcag 2640 gtgatccact tgcctcagcc tcctgagtag gtagtactac aaatgtgagc caccacacct 2700 ggctaagttt taaaaaattc ttttgtaaag atgggatctc actatgttgc tcaggctggt 2760 cttgaactcc tggcctcaat ccttctccca tcttagcctc ctgagtagct ggaactacag 2820 gtgtgctacc acggggcctg gctataaaat cagaattttt tttttttttt tgagttggag 2880 tcttgttctg tcgccaggct ggagtgcagt ggtgcggtct cggctcactg caacctccac 2940 ctcccgagtt caagcgattc tcctgcctca acctcccgag cagctgggac tacaggcctg 3000 cgccaccatg cccagctaat ttttgtattt ttagtagaga ctgagtttca ccatgttggc 3060 caggctggtc tcgaattcct gcccttgtga tccacccgcc tcggcctccc aaagtgctgg 3120 gattacaagc gtgagccacc acgcccggcc catatatata tattttttaa tcttttcttt 3180 tagagacagg ttttcactca gtcacccagg ctggactaca gtagcatgat catagctcac 3240 tataacctta aaccctggac tcaggtgatc ctcctgcctc agcctcctga gtagctagga 3300 ctacaagcac gcaccaccac acccagctaa tattttaaaa cattttttgt agagacggga 3360 tctcactatg ttgtccaggc tggtcttgaa ctcccaacct caagggatcc tcccacttca 3420 gcctcctgaa gtgctgggat tacaggcgtg agccaggagc ctggcctaaa ctctgctgtt 3480 taatcctaac aacaggcctt cctctgccaa cacctacccc cctgtcctga cttgctgagt 3540 cctataacag gtctgctttt tgagccctgt tactgctgct catcactgtt ggcagagcca 3600 gtctcagctg ggcaatgccc ttagcctcag gggctgggtt gccttggcca ctggtggggg 3660 cggacttgag actctggtgc ctcccatgtg gaagacccaa gtctctagtc tgtgaaggga 3720 tcctcaggat ttcaaaaagt cctttgcctt tgattagggg attattgctg ctaccaaagt 3780 ttttgggtca ctttcctttg gtctggagtc gtgtgggcac cattcatgat gagggtactg 3840 ccgcatgtgt ttgattaatg gtgtatcaaa tctttcaaaa tatagggctc agggtgtagt 3900 gctatgagca gctcttgaaa gtgaagcagt tggcagcctt ggggtgttct gaggtctctt 3960 cttgctctga ggccagcaag gccggatgta tgtaggcctg gtctttggcc cacaccttat 4020 cttggccact gttagaggcc aggtcgaagg gcagtgtcac agaggtcaag tttgaaacat 4080 catgagcagc ttgggctttt cggattgagt tagtgtggcc ttggtccctg cctccaaccc 4140 ctacatgtga ctgtcactgg gttgttcaaa tctagattca agatcaggtt ccgcccagaa 4200 tatcttgggt ggagttgaag agtctgggaa gtggaagaga gagccagagt atgttgagcc 4260 tgctagttac gggaaggaaa tttttttttt ctagaaatct ccagcatgct cagtttgggc 4320 atgtgtctat gattttaata tcgggatctt agctgtgcca gttactagta gtgtagctgg 4380 ggcaagtcac ttaattgttc tgtgcctcag tgtccttgtc tgtaaaggct gggtaattcc 4440 tacctcatag tgttgttctt agctttaagt aagttaatac aggtaaagca ctagaacagt 4500 gcctggcata cagtaagtgc ataataaata ctggctgctg ctgttatggt ggtggtgatt 4560 ttccttccca tttctgtgca gtattcatct gagcagggag gtaggagact cagatattga 4620 ttatgaaaat taattgaaat cagatgagga tattagcttc cttcatatac agtttgcctt 4680 tgtttctgga ggtggctagg actgagcaga tttgcacctg ggcttcagcc cctgtgagct 4740 acttcatctt tttttttttt ttttttgaga cagggtctca ctttgtcacc caggctggag 4800 tgcagtgatg ccatctcagc tcactgcagg ctcaacctcc tggattcaag cgatcctcct 4860 gcctcagccc cctaagtagc tgggactaca ggtgtgcacc accacacctg gctaattttt 4920 gtatttttag tagagacagg gttttgccat gttgcccaag ctggtctcga actcctgagc 4980 gcaaatgatc cacctgcctg ggcctcctaa agtgctagga ttacaggcat gagccactgc 5040 gcctggctgg ctgattcatc ttattcccag cccttggtgg tcccagtctg cagtgagcct 5100 tggactctga tttctgtctc cctgccctgt gaggctgaca aaggctccca gccttgttct 5160 gggattggca gatgcctctg gagagaacat cctagatatc aggcttatct ctctgggccc 5220 ccttccccct tctcctggac cttgacttct taatcctctg cttgattggc tctccagcat 5280 cttttagcag atctccccgc tgccccatta tttggactgc tttcctggct gtttgcagtg 5340 ggaggattgg tctgagtggc cccacctgcc attaccgaag cagctgtccc agctcgttct 5400 gttcctgtgt gagcttggcc acctttctgt cctccttgct tgagtttcat tggctgtaaa 5460 atgggtaatt agagaaaaac tcataataat ataagctggc atttagtatg tttttcctaa 5520 gatccaggca ttgggctgag tacttacatt gtttcaatta atcctcccaa tagccctgtg 5580 agctggggac agttgtatct attttgtgtc caagaaaact gaggtgaagg acacagttgc 5640 acagctggga gttgggactg gaaccctggt ctgtactgtc ctctgaaaag gatgaaggga 5700 agtagtaagt attagaatgc ttcaaaaagc tccaagcagg caagactcgg gtgacaggac 5760 ttggggtggc aagacttggg tggagtggaa gaggaaggaa tctatcctgt gggatagttc 5820 cagggatggc ttctggtggg gctgggctga agaggtgcat tctcaacctc atttgttttc 5880 cctgcaggcc accctgaggc acaaactaac tgtcatgtac agtcagatca acggggcgtc 5940 gagagcactg gatgatgtga gaaacaggca gcaggatgtg cgggtgagtg gcagggctct 6000 ccaaggccag ccctgccctg tggcttcgct cagcttccac gcgggatggg cgtggctctg 6060 accatgacaa ggctggtgtt gggtgcactc acagtgtcag gatgaaacat caaccttgca 6120 aagcatgcag acagctgtgt gtctgagaga gatcctttgc aaacccaaac caaaaattac 6180 ctataagccc aactgtgggg gaaatcatta taaattagga ttaaaaagta gtaattcaga 6240 aaaatggtta tgatataaag tgggagaaaa agaggaagca aaagtatata gctcataggg 6300 ttgcatctat ttacaaaaag gaagtatatt agtttcatag ggctactgtg acaaattgcc 6360 agaaacttgg tggcttacaa caacagaaat gtattctggc agagttctgg aggccagaag 6420 tccaaaatca agatgttggc gtggccacac tcccgaagtc tctaggggag aacccttctt 6480 tgcttcttac agtttctggt ggctcaggca ttccttggct tttgactgca tcactctgat 6540 ctctgcctct gtcttcacat acctgtctct tcttccctta gtgtcttcca tctccaataa 6600 gaacccttgt cattggattt agggagcacc taggtaatct atgatgatcg aatctcaaga 6660 tctttaattg catctgcaaa gacctctttc ctaagtaaga ttacagtcac aggttctggt 6720 atatggacat gtcttttggg ggcccagcat tcaacccatt acagagagca tggaaagaaa 6780 ctgaaaggga atattataga aggatcatag tggaagtgtt ggagtggtaa attaggaatg 6840 gttttgttcc ccctgttttt cagattttcg atacgtttaa attgctttta aatggaaaga 6900 tcagattttt accctttttc agagctgcag caccagtttt attgcagcag ttatggtttc 6960 attagatgag atctaggtta gaataccagc tctgtcacat actagtttat gttgctttgg 7020 tcatgttgac agatctttga accttcgttt cccaatttgc acaatggaat cacaagttct 7080 ttctgaagat gactgtgaga caaatgaaag aatgcacgtg aaagtacctg tatttgttag 7140 agggcaggct atactgctgt aacaaagata caccagaatt cagaggctta aatatgattg 7200 aagtttattt taccaactga cagtccaaac ccacacatga gtaacccaga ctaagggttc 7260 atgtgatcat tcagggatta agggttctcc cttcttatta ccctactatt ccacaaggta 7320 ttgtcccagc ctctttggtt gaagctgggt taccattaca tctttgttct agcctgtgac 7380 aaagggacaa aaaggaggtg gaggacaaca agcaattttc ctttacacaa gtgacacagt 7440 gttgcacata tcactcctgg gcatgggtca ctggaaggag ttttgtcaca tcgcctagtc 7500 tagctgcagg ggaggctggg aaatgtagtc tgagtagcca cacgcccaga tgaaactcca 7560 gggatgttct cttaccaaaa ggaaaaaagg gaaaatgaac actgggggac acttgtagtc 7620 acattagaag ttgcacagtg aaagattggc aaagaaggct gggcgcaatg gctcacgcct 7680 gtaatcccag cactttggga ggccgagacg catggatcac ctgaggtcag cagttcaaga 7740 ccagcctggc caacatggcg aaaccccatc tctactaaaa atacaaaaaa ttagccgggc 7800 gtagtggtgc atgcctatag ttccagctac tcaggaggct gaggtaggat aattacttga 7860 acccgggagg cagaggttgc agtgagccga ggtcatgtca ttgcacttca gcctgggtga 7920 caagagtgaa acttcatctc aaaaaaaaaa aaatattggc gaagaaatac ttgaattcct 7980 cttttccagt ctactgtcct tcttgtgtgt ttttctcttt gttatgtttg attctgagct 8040 attgtcttag tctgttttct gctgctataa cggaatacca cagactgggt atttgcaaag 8100 aagagaagtt tatttggctc aaaggttctg gagcctgaga ggtccaagag catgcttcca 8160 gcatctggtg aggatcatcc atagcggaag gatggaaggc aggagagcgg gacaggaaga 8220 aggaggtcaa acccctgtga taagaatatg aatccattca tgagggcaga gccctcgtga 8280 tctaatcacc tcagtggcaa ttaaatctca gcatgagctt tggaggagac attcaaacca 8340 tagcagctat tgaggctacc caagatgtga aacaaaattg cagctactaa agcagtgctg 8400 tccagcagaa atacagtagg agtgcagatg tcattttaag ttaatttgaa tagtacatga 8460 gatttattgt attttattta tctatctttt ttaggacagg tctcactcta tcacccaggc 8520 tggagtgcag tggaacaatc atagctcact gcagctttga actcctgggc tcaagtaatc 8580 ttcctgactg agcctactcc ccttcaccac ccccactctc caggaataca gtccatctaa 8640 tttttttatt tttatttttg tagagaaaca gtctgtctat gttgcccagg ctggtcttga 8700 actcctggcc tcaagcgatc ctcctgcctc ggtctcccaa ggtgctggga ttatgagcat 8760 gaatcactgc acttgactga gattaatttt aatattatag tttatttaat ttggtatatc 8820 ttaaatatta ttctttcaac atatcatcaa tttaaaagtt ttaagatatt ttacattcct 8880 ttttttggtg tgaaaccctt gaaagccagt atgtatttta cactcatggc aagtctcagt 8940 ttggactaac cacatttcaa gtgctcagta gccacatgta gcaagtggct atgttattgg 9000 aaagtacaat cctagagcat gatattctct agaaattttc cagagctgca tcctgacttt 9060 ttgttttgtt ttattttatt ttattttttt gagatagcgt ctcgctgtgt ctcccaggct 9120 ggagtgcagt ggcacgatct cggctcactg taacctctgc ctcctgggtt caagcgattc 9180 tcctccctca gcctcctgag tagctgggac tacaggtgcg cgctgccaca accagctaat 9240 ttttgtattt ttagtagaga tggggtttca ccatgttggc cagaatggtc ttgatctctt 9300 gctgactttt atttttaaaa attgcccatg aaccaccttt agcttaagaa ataaaatatc 9360 atcgatgcag cccctcccat ctgtttctcc cttattctat ccctcttcct ctcttccaga 9420 agtaaccact ctcctgaatt tatttattca ttcccatgcg tttctttgtg catatagcca 9480 ttctgtttgt ttctaagctt tattgaaaga gcatcatatg gtggatcttc ttctgcaagt 9540 tgtttttctc tcatggagat ttatccaggc atagctccag tttattaatt ttcataactg 9600 tttaatattc catcctatga atagctccca gtctttgctt caagtcttct tctgatggat 9660 gttcaggttg attccaggtt tttgctactt cagacaatgc tgcctgtgaa cattcttatg 9720 catgtctcag gggcaggtgg ctgcaatttt ttgtagcaca tattcctagg actggagttg 9780 ccgagtcata gcctatgagc acctccaaat ttattagata ttgccaagtt gctccccaca 9840 gtggtttgtt ctcccacaaa gtacaggtaa gagttcctgg aactccacat ctcacccatg 9900 cttggggctg cagccttagc acttttgccc ctttgacggg catcctctta ccttggccct 9960 ctcagatgac tgcaaacaga aaggtggagc agctacaaca agaatacacg gaaatgaagg 10020 ctctcttgga cgcctcagag accacctcga caaggaagat aaaggaagag gagaagaggg 10080 tcaacagcaa gtttgacacc atttatcaga ttctcctcaa gaagaagagt gagatccaga 10140 ccttgaagga ggagattgaa cagagcctga ccaagaggga tgagttcgag tttctggagg 10200 taggtcgcca caaggcattg ttgaatgttt tttttttttt tttgcctgat gtcatgctgt 10260 tgatgatagg gccctcaagg agcgggagac tggaagaggt cactggaagc atccctgaga 10320 cacacgctgt attagttttc tgagctgggc aacaacttac cacaaatgta ggggcttcaa 10380 aaaacacata tttattctct cagagtttct ctggctcagg aatctggaca tggctttatg 10440 ggtggcctgc atagggtctc accaggctac agtctgcgag ggatggccag gctgtttcct 10500 ttggagctca gggttgtctt ccaggctcat gtggttgttg gcagaattta gttccttatg 10560 gttgtaggac tgaggccccc cgcaatttcc tgctggctgt ctctcagatt gatagctgac 10620 ttcttcaagg ccagcatgag aatttctcct ctagaaagat gcaatccctc ttttaaggtc 10680 ttttacctga ttaagtcagg cccaccccag ataattgccc tttgatgaac tcaaaatcaa 10740 ttgatttgag atgctaatta caccagcaat attccacccc ctttgccaca tgacataacc 10800 taatcacagg agtgatatcc tatttcattc accgtcctgc cgatactgaa ggggagggca 10860 tactataggg tatgtacatc agtcggggtc gggggcttga gggccatctt agaacgttgc 10920 ctgccactca cactccacct cttcctccca tccttcctcc agcagctcct ggaagtgtgt 10980 gttagtagtc ccttttgcat tgctataaag gagtacctaa gactgagtta tttatttatt 11040 ttatgtattt attttgagat ggagtttcac tcttgttgct caggctggag tgtagtggtg 11100 tgatcttgct cactgaaacc tctgccccca ggctcaagtg atcctcctgc ctcagcctcc 11160 tgagtagctg ggattacagg cgcacgccac catgcccagc taattttgta tttttagtag 11220 agacgagatt tcaccatgtt ggccaggctg gtctcgaact cctgacctca ggtgatccac 11280 ccacctcggc ctcccaaagt gctgagatta caggtgtgag ccactgcgcc caaccagact 11340 gagtaattta taaagaaaag aggtttattt ggctcacagt tctacaggtt gtacaagcat 11400 ggcaccagca tctgtttggc ttttagttag gcctttggtg aggaaggaag cttttactca 11460 tggcagaagg tgaagctggg agcaggcatg tcacagggtt agagagggaa caagagacag 11520 aggaggaagt gccagcctcc gttaaacaac cagtttgtgc aagaactcac ctgttaccat 11580 ggggagggca ccaagcggtg catggggaat ccacccccac gacacgaaca cctcccaccc 11640 tgccccacct ccaacactgg gaatcacatt ccaacatgag atttggaggg gacaaacatc 11700 cagactgtat caaagtatct gtctaaagca tgggccccct gctgtttctg gcctcatgtc 11760 ccttaacatg gcctgcaggg tcctaggtga ctgggccccg cctgtgccag cagtctcatc 11820 tcatatcatc catccctggc tcatgcccta tgcactcact gttccccaga cacagtgggt 11880 gtgggtggga tgccatgctg ggcattcttg cctagtgagt cgtatccttc cagggccttg 11940 gatcccattg ccatgccgta tcggtgttcc caagcatgcc acactgtttc tacagtaaca 12000 ttgggtgatg ttgtttcaga tgtgcatttt ttcatggctg ttttgactgt aagcccatct 12060 cttattaccc ccattcatcc tcctgccaat ctgaccagca catagcagag tgcttggcat 12120 atagtggtgt gccccattga ttgagtgacc tgcagctata cctgaaagac ccagattcct 12180 tgttcagatc agggggtggg gagacagtgc aggagtgaat gggaaccttg tctctgtgca 12240 ctgaggcctg atgcgtgcct agtctggatg gcgttaagtt tcatcccaag tgttcattcc 12300 tgtctgttgg agcagctgcc ccgagctctc gaaggatctg agtgcacatc taggcttctt 12360 gatactgtac aaccctattt ttctcaaagt ttgggacttc caggaagacc ctcccttccc 12420 ataagatatt ccctggctgc aatccacatc ccttgagatg tccctgtctg ttaccagaat 12480 gtgtgattag agctgttccc ctaggattgg ggtggtcctg agtttcctgt gcttttccac 12540 ctttagctgg aaaagtggaa agaacattgt cctgggggat cggaagatct cagtcccagc 12600 tgtgtctgtg cccctagctg gtggagttcc tgccatgtct agacctcagt ttccccatct 12660 gtaagatgag tgacctctgg ggtcccttcc agctccagat tactgtgtct tcaagtgtca 12720 tttaaactcc tggtgacctc tggcctgtaa tttgagaagg ccaattggac ttagcactct 12780 tgggcatagg ggagagcctc ctggggttgg tgaggactga gcccaggtcc ccgtttcacc 12840 agagctgcct gcactaaccc cctggcatgt tccagaaagc atcaaaactg cgaggaatct 12900 caacaaagcc agtctacatc cccgaggtgg aactgaacca caagctgata aaaggcatcc 12960 accagagcac catagacctc aaaaacgagc tgaagcagtg catcgggcgg ctccaggagc 13020 tcacccccag ttcaggtgac cttagcagcc ccctatgctg ccccctgtgg aaccctgcaa 13080 cccgatgggt cccctaaatg ttttgggatg ggagattggg gcgtgatccc gagcactggg 13140 gcctgagttt cagggctcct gaaacatccg ccctctgtct ggaggctggt ggggagtctg 13200 cttcatcctg acattttagg atctttccta aagcattttt ggggacttga tgttaatgct 13260 tggactttct ggacagatag gctcttttgg cctgtgatct tgaggccaac ttggatgggt 13320 tttgaataag ctagggcttg ttattgtgtt tggtggggga atggcccgtt tgggggagaa 13380 atctagaatt cctctttgac accccctttt catcacccca tatccaatcc attgccaaat 13440 ttcacttctt tcaatctcct gactcagtct gctgatccta gcactgcttg cctgttctcc 13500 attgccatca cctgtcgccc catgactgta gcagcttcct aaaccctctg catctgccgc 13560 tgcagccaga gagggatttt catgatgcag agctcatgtc ctgggaccta ttctgctttg 13620 ccctctcact aggcttccta ttactccctg atgcagacca aacccttgac cctggtctag 13680 atgccaggtc aagccctgtt ggtctctctc cagccccaag gcgcccccca ctctcctctg 13740 gtgcatctct ctgcattcag ttcctcaaat atgcaaaact tcctgctaca gggcctttgt 13800 gtttgctctt ccccactctc cagaaggctt gaactccacc gtcttcacta attaactcct 13860 ctcacccttc aaagatcagc aaaacattac atttatagcc agcctcccct gccactgctc 13920 ccactcctca gactgggccc agggtccttg tcatcctctt gggactcttg catgggtagt 13980 atcgcattct tccttgggat ccccgtatat ctccctcatt aggttgtgat ctctatgagg 14040 tcaggggctg cggcttattt atttatttat tcaggatctt gctctgtcgc ccaggccgga 14100 atgcaatggc gtgattatag ttcactgtaa cctcaaactc ctgggattaa gtgatccctc 14160 tgcctcagcc tcccgactag ctaggactat agatacacac catcatgccc ggctaattaa 14220 aaaaaaattt tttttttttt gagacggagt ctcgctctgt cacccaggct ggagtgcaat 14280 ggcccaatct ctgctcactg caacctctgc cacccaggtt caagcatttc tcctgcctca 14340 gcctcctgag tagctgggat tacaggcatg tgccaccaca cctggctaat ttttgtattt 14400 ttaatagaga catggtttca ctactttggt caggctagtc ttgaactctc gacctcaagt 14460 aatctgcctg cctcagcctc ccaaaatgct gggattacag gcgtgagcca ccgggcccag 14520 cctaaaaaat tttttttgta gagccagggt ctcgctaagt tgtccagact gatcttgagc 14580 tcctggcttc aagtgatgct cctgccttgg cctcccaaat cattgggatt acaggcttga 14640 gccactgtgc ctggcctgtc tgatttttct tcaacgctat ttccctagtg cctagcacag 14700 agcctggccc atagtaaatc tctgttgtgt agtccaagat tgggcttgaa tctcagcttg 14760 gcctgccgag tgtccttgca gaagggcttc tgaaatcggt ggcggtgcag ccccattgtg 14820 gaagtgctgc gtgggggcaa agctataggt ggctggtatg tgggaggagg ggagagaaag 14880 aatagtggga aatgaacaag tgttgaccca gcctccgcta catgaccttg atctcattca 14940 ggagtcacat caaccatgag caaggtgaag tcatccccat tgtgtgctcg aagaaaccaa 15000 agctcagaga agtctcagag gccacagagc tgggtgctgg tggaaaaaag acttggccct 15060 tgctacattt taaaaagtca gagaaaaaag ggaatacacc aaactggttc ttagatcacc 15120 ttgtttgtag ctcttgtaag cgatgggatg gggaaggtcg gaggaaatgg ctgtctgtgt 15180 aaaccctctg cctgcccacc gacccatggc aaagctggct gctagggtca gtgagaggag 15240 ccgcacacac tgagcccttc ttctctgaca caggtgaccc tggagagcat gacccagcgt 15300 ccacacacaa atccacacgc cctgtgaaga aggtctccag taagtagccc cacccctgtc 15360 tccatggcaa cagcatggct gtggcctccc caggctctgg ccaagtcact tccggcccat 15420 cccgagtcac tgtggggcct ggacctcagt gggaaagaag ttggtggcag tccccaccgc 15480 ggggggctgt agggctgtgg cagaagctcc cctgtccatc agcattgcac gtttgggctg 15540 cctggaacag caagatgcct gaggctggct tgttttgaga aatatttact atacccaagt 15600 ccttgggcag agccttgggc aaggagactg atggcggtcc aggaagggtg gaggctaaac 15660 ctccttggca tctcagcagt gttaagtctc tgggttgtaa tgggcttctg ctattaccat 15720 gtttctcaac tgtttttttt tttttggttt ctcactgtta ccctcctcct gctcatcccc 15780 agaaccattt gagaaaattt ttttcctaat tacaacccat gaaattttaa taccacccat 15840 ctactgcata actgtttatg tttgtgcttt tacacataaa aagagtaatc ttttttactc 15900 cccctccccc aaggatccat ttttgccctc ctaggggtac tatccccctc attgggcagg 15960 cacctgccat tgacatgggg gttgaggaga agagtggtcc tgttctgcaa atgtggacaa 16020 tagtgatatc gaggccacag gatgattctg agtgatgagg ggacactgat tgtgagagct 16080 ctttatgagc catataccac tgagatgtcc atataccact gagatgtcca tataccactg 16140 agatgtccat ataccgctga catgtccata taccgctgag atgttcatat actgctgaca 16200 tgtccatata ccgctgagat gtccatatac tgctgacatg tccatatacc gctgacatgt 16260 tcatatatcg ctgagatgtc catataccgc tgacatgccc atataccgct gacatgttca 16320 tataccactg acatgtccat ataccactga aatgttagct gtcattgctt ccctgaaaag 16380 ttggtgtgaa cccaggaggg ctggctgcac actgctggtg ttggggggat gagagagcca 16440 gggcttgctc tttgttgttg ttgcttgtgc ttagtgttca ggtcacctgt agcaggcaga 16500 gtgtggtatc ggtccccgaa tggaacagag tcagggctgg gatgcctggt gcagatggag 16560 gagggagccc ttagctgggc cggaaaacct cgaagggtac tttgtgtgaa aagaggagtg 16620 aaagaggaag tggaaaggta ctgttgacct gtgtccttcc ccagtcacct tcctctgaaa 16680 gctcctccct tagtatgaat gaggtaggga tgccggccag caggcaggtg tgttccccag 16740 tgaggtgagg tcctcatgtc acagcagaat gacaccctgg agaataaagt tcgggaatga 16800 atcaaggctc ctctaggctg aaatggggaa ataggggagg gggtatggag tagtcccagt 16860 gcagagagat gtctaagggt aggtgatgag ggaggtgcct gtgccctaga ttgatgtcac 16920 agcctgggcc tcccaagatg ctggtccaca gggagtgggt gggaaagtgg cagaagggga 16980 ggaaatggga gagtgagcga ctgtgagtga tgtgacttcg aagtaggaga agcggtagca 17040 agggggcttt gtggtgtgat gggggtctgg gggagggaga gggtaatagg agagagcagg 17100 gcttatctgt aatgtcaggt gtcctgcagg tgggcaggga tatgatttca aaaacgcccc 17160 tttttgttcc agtgttgtgg aaggtggcaa agaaggaagc tgggatcttc tagcccactt 17220 gagtggactt tggactcacc tggagggctt gtacatcaca gattgctggg tctcaccccc 17280 agcctttatg attcagtggc tctggcttgg ggcccaagaa tttgcattta ctatttttta 17340 agagacagag tctcgctctt ttgcctaggc tggagtgcag tggtgcgaac agaactcatt 17400 gcagcctcga actcctgggc tcaagtgatc ctcctacctt agcctcttga ggagttagga 17460 ctgtaggcat gtgccactaa gccaggctaa ttaaaacaat tttttttttt ttgtagagac 17520 aggatattga tatgttgtcc aggctgatct tgaactcctg gcctcaagca atcctcctac 17580 ctcagcctcc caaattgctg ggattacagg agtaagctac agtgcctgac caataatttg 17640 catttcttag gggttctctg gtgatgtcac tgcagctggc ttgggcacca gactttgaga 17700 acgactattt tagccaaact cagttcctgt aggtagacac tccagatatt tttaaaagac 17760 ttggcaagca tttttttttt tttttgagac agggtctcat tttgttgctg aggctggagt 17820 gcagtggcaa gaacacagct cactgcagcc tcatcctcct gggctcaagc aattcttctg 17880 cctcacctcc caagtagctg ggactacagg tgtgtgccac taggcctggc tagttttttc 17940 tttttttttt tttttgtaat tttgtagaga tggggtttcc ccatgttgcc catgctagtt 18000 tctaacgctt gagatcaagt ggttcctgca ccttggcctc ccaaattgct gggattccag 18060 gcatgagcca ccatgcgccc agcccttggc aagcattttt tttttttctg attccctatt 18120 tggtttttgt gttttttcct ttatggaaat ataatttacc tgcagtaaat ctcttttttc 18180 cctgcagtaa atcttaaatg tgcagttaga tctattttga caaatgcata catgtgttac 18240 cattgttcaa atcaagataa agaatttccc tttctccaga aaattccttc atgctcctta 18300 ttagtcagtc cccgccccca cgaccgctca tgctggtttc tatcactata gattcatttt 18360 gcctgctgac tccttctgtt tttcaaatta aaacatcctt ttatttgtct gggtcaaatt 18420 catgcacatg aatgttatta ttttgaaaat gtcattgtgt tgtgccttaa aagaaattaa 18480 atctgaaaac tttcttttgc agaagaggaa aagaaatcca agaaacctcg taagttatgc 18540 atttctgcaa ctgttcgttt cttgagactg cccgagcaca gaactaaatg ctggtagtga 18600 gagtcgacct ctctcatcag ggttctgtga tgggttctat atgattccat tctgtaatgg 18660 cagcaggcct gaggctggag gggaggacaa gcataagtga gatgaataag gtgactttag 18720 ccttgtctgt tttcccctgt gttgtcctgc tgttcaggcg tgtgtttgta gaagtggctc 18780 ttaatcagtg tggaagggag tagaggccat cccgttgctc tgtatcatat ttccctttcc 18840 ttacccccag cccctgtccc tgccttaccc agcaagcttc ccacgtttgg agccccggaa 18900 cagttagtgg atttaaaaca agctggcttg gagggtaagt tatcatgggc aaggagcttt 18960 ggggtgtcca ctctgagttt gttctgcaaa tcactctctg tttcctctcc cagctgcagc 19020 caaagccacc agctcacatc cgaactcaac atctctcaag gccaaggtgc tggagacctt 19080 cctggccaag tccagacctg agctcctgga gtgtgagtag cttcggggct ggtgcgcggt 19140 tccactggtt ctgtctctcc tgcgccgctc tgtgcagctc tctgtgaggc cagctggccg 19200 gctatatcag ccactcccct tcacttgtcc tctactttgt tgctccctta ttttctccca 19260 gtcatcagtg cctttcttta cttttcctaa ctagctcaga tttcacaatc ggtcctttga 19320 ctctccctct tgccgtctag tctggcccaa ttgagctcac ctttctctgt gggtcggcgc 19380 ctgcatgtcc tctgcagaga aggcagggcg attgtggtca ccggccttca ccgggcgtcc 19440 cgttgccggc atgcctgccc tgcatctcct gtctcctccc caccactctc cagagccacc 19500 attcccaaga cttcccactg gccatagctc catccttctc cctgtcccgc ccaccttctg 19560 cttcacgaaa aagacagaca ctgtagaagg ggctccccct gctctgttat tgacatcgca 19620 tctgcatcta ctgtgccccc ttcctcgcct cctggcccct ttcccaagac cccctggccc 19680 tgggctctgg agtctgctcc ccacacgctc tctggaatct tctgctctct cttaggcctt 19740 tccattccca tctcttcacc ctcagcatct ctacctaggc ttgcctatct gtagcacgtt 19800 tacatctctc caaatcacaa aaacaaacct agcaaaactg tctcagctgc agctctgttt 19860 cctgcctccc tttcacccga atgcttgtta aaaatacaaa ttactagctg ggtgcggtgg 19920 ctcatgcctg taatcccagc actttgggag cccgaggcag gtggatcacc tgaggtcagg 19980 agttcaagac cagcctggcc aacatggcaa aaccccatct ctactaaaaa tacaaaaaat 20040 tagccgggcg tggtggcaca tgcctataat cccagctact tgggaggctg aggcacgaga 20100 atcacttgaa cttgggaggt ggaggttgca gtgagccgag attgtgccac tgcactccag 20160 tctgggcaac agagcgagac tctgtcttaa aaaacaaaca aacaaacaaa aaaccccaaa 20220 aaccaaattc ctgggccctg ttctagccct acaagatcag actcaggcaa gcacggaaag 20280 ctgtatgttt agctcatttc ctccatgatc cttttgagga cagttgggga aaagcagctc 20340 tgcagatttt ctacattggt ggctctcaac tgtggctgcg tgttggaatc ctggggagct 20400 ttttaaaatc cccgatgccc aggctgtccc ccagactggt tcattcagaa tgttggtgtt 20460 tggctgccca gggttgcaat gtggggccga ggtgggagcc tgaagtctaa tcctcgtgcc 20520 actgcattct gacctctgtc ctgcccttga cccaggcagc tctgaggtca tcagtgtact 20580 ccgtgtcaca aaatccaggg acgcctttca gcttttattt agcttcacct gtcagtggca 20640 ttcctcctcc ttctgacact ctctcccctt gccgcttgag ctgctatacc cccctgggtt 20700 tccatccacc cctcctttgc aagctcgtcc aacctggaag tgggtggagt tccgttgtca 20760 aggccaggcc tagggccctc ccactttccg tcttcttgtt tcttctgcac ccacgtgcaa 20820 cactccaggc agcacctgcc agtggtaccc acttccatgt ggaggccaaa gccaatcact 20880 gaaaaatata gcccttcctt tatcataagc agctgcagca ggcttggtgg ggatctgtgg 20940 gtcccttgct ttttcagggc tggccttctt gtagccctcc gctgtccctg ccccccaaga 21000 gaggcccaga ccagccctgg gctgcagcta actcttgccc aagctgctag cggcggtgct 21060 gagccacgtc tttcaggagc tgctgtgcac tctgtggcag ccgcataaac agggaaagta 21120 aaattctatc aacttagggc cttttccacc cccagcttat tcattttgcc ttgggggaaa 21180 ctcgggctcc agtgaaaagt gtcatgccct gagccaccta gcaaatgagt gcctcgcttg 21240 gctccctgct gcccaccagt taaagtctag cctcctaagc ccttcccttt ctagccccaa 21300 catgtttccc ttctcctgct gcatctgtgt tcccagccac ccagcttccc cgatcccagc 21360 acatgcgaat ccccttggca ctccgtggct tcactctcac tgttccctct gtctggaaca 21420 gcttttctct ctctgttctc ctctgtctag ttctcatctt ctgttacttg gctccaattc 21480 cagcctgcca gtgaaatgtt ccccaactgt agcatcagtt gccttgtctc tgtgtcccta 21540 cagtgtgtgc ttaccctttt ggcagagtgt gagtctcatg gtgttgttac tgatggactt 21600 gccttgcttg ttggagtgtg aactccatga ggccaggcac cttgtctctc tggttcatgg 21660 ttgtgcatct tgtgccagaa tctggtgtca gtcaggtgcc tggtaaatgg ttttggatgg 21720 atgaatggac agacatacgg atgactgata gatgatagat ggctggctgg ctggacagaa 21780 tggatggatg aatggatgga tagatgagct gatgaataat ggatgggtgg atagatgggt 21840 ggatggataa atggacagat ggatagataa gtggatagat taatgaatga atgaatggat 21900 aaatggaaag gcagaaggat ggatagataa gtggatagat ggatgaatgg aagaatggat 21960 agataggtag atagatgggt atatggatac atgggtagat ggatagatgg atggatggat 22020 acatggatgg attgatggat acatggatag acagatagat ggatgaatag acagacagat 22080 ggatagatga gtggatggat ggaccaatgg gtagatgggt gcatggatgg ataaaactag 22140 tgaataaata gtaccacttt ggcaaaggtc taaaagagca ctttgggccc cttgtaatta 22200 attccttggg gtgatgtttt ctcagattac attaaagtca tcctggacta caacaccgcc 22260 cacaacaaag tggctctgtc agagtgctat acagtagctt ctgtggctga gatgcctcag 22320 aactaccggc cgcatcccca gaggttcaca tactgctctc aggtgctggg cctgcactgc 22380 tacaagaagg ggatccacta ctgggaggtg gagctgcaga agaacaactt ctgtggggta 22440 ggcatctgct acggaagcat gaaccggcag ggcccagaaa gcaggctcgg ccgcaacagc 22500 gcctcctggt gcgtggagtg gttcaacacc aagatctctg cctggcacaa taacgtggag 22560 aaaaccctgc cctccaccaa ggccacgcgg gtgggcgtgc ttctcaactg tgaccacggc 22620 tttgtcatct tcttcgctgt tgccgacaag gtccacctga tgtataagtt cagggtggac 22680 tttactgagg ctttgtaccc ggctttctgg gtattttctg ctggtgccac actctccatc 22740 tgctccccca agtaggcagg ctgtaggcac ttgggctgac tgcctgcaga agtcccaaga 22800 ccctagtgaa aatacagcag gcagaactct ccttggataa ttccccaaga ggtcccaagg 22860 attgggagca tgggagggga gctggcggga gggtgggagg tgggatttag ccaggaaagg 22920 ggtgagagtg attgtgttgt gggcgaggag gcgtttccac cccctggtgc ctatcagggc 22980 agggtgacct actccccatt gttctggaaa tctccaggct gctgggcagc tgggcagagc 23040 tctgggaagt gaagtcatga gtgcccgatt cctcttagag aaaatccata gctactgtag 23100 gttctgtctt gggccacttg gatctgaagg ctgccccttt gctctctggg gtagccttca 23160 gatcttggtg ttttgaattc ttactataga tgtttttaaa gttccaaagt cattgagttt 23220 caatgttaca taaactccat tacccgcatg ttggggcttg atctcctggt tattatctgt 23280 gcttgaggaa acacccacag cagtctctac ccagaacagt ttcctaaaga ggcataccct 23340 cttcctccac tggaaaatag tgcgttccct tcctaccctg cacacccatc gcccccacat 23400 tgatggtttt caaacagcaa acttttcagc taaagcttca aaccatgatt ggaatcagcc 23460 tgtgttggat ttgtgattca gggtcatggt gaccctgatc cagtttgggt ggaaatcctt 23520 cctaagtatc ataagaagca tcttggcaga gatgctttgg tggcagccat gagctttgct 23580 ggaggccttg cttcccatag ccttggctgt ggggcaagga actctgccag gcgaggggga 23640 tgctgccctg gatcaacaga agcctggtgg gtttgctcgt gttagagtgt cctgccttct 23700 tactgacaac tcttctcggt gatagcctct cttccctgga ttgtgacata tggaatgaca 23760 gtgcaggtac caccgaggct agcacagtca agcctccagc taagctggat ccctgaagcc 23820 tgctatcatg cagacaggct atgcggctgc ctcggaccat gctaggccac ttgctggggt 23880 gtcaacctac caccaaaggg gtcttttagc aaacctcatg gggaacagga acattcctgt 23940 tcatccctgg ccacaggctg cagacccagc actggccctt gcgtgagtca gagcctgggg 24000 ctggccctag ccccttctac tgacttcctc atttaagcca attatataag ctcacattga 24060 tcagggaggg agggaaagag ctaaagaggg tcacacaagt ggctattttc cctgcagtgt 24120 ttctgtgtgg tgaaaataac ccagtccact aaggggcggg agtgaatgga tggctggatt 24180 ttccccaagc tccttatagc ctaatgttgt caggatgtga gtatgaggaa tttagcctct 24240 tatagtgaaa tgagtccaac tctgggcttt gcttagagga gagctcccgt caggc 24295 5 20 DNA Artificial PCR Primer 5 ggcgtgcttc tcaactgtga 20 6 18 DNA Artificial PCR Primer 6 tggaccttgt cggcaaca 18 7 22 DNA Artificial PCR Probe 7 cacggctttg tcatcttctt cg 22 8 19 DNA Artificial PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial PCR Probe 10 caagcttccc gttctcagcc 20 11 20 DNA Homo sapiens 11 agggagtaga ggccatcccg 20 12 0 DNA Homo sapiens 12 000 13 998 DNA Homo sapiens misc_feature (667)..(667) n is a, c, g, or t 13 gggtgagagt gattgtgttg tgggcgagga ggcgtttcca ccccctggtg cctatcaggg 60 cagggtgacc tactccccat tgttctggaa atctccaggc tgctgggcag ctgggcagag 120 ctctgggaag tgaagtcatg agtgcccgat tcctcttaga gaaaatccat agctactgta 180 ggttctgtct tgggccactt ggatctgaag gctgcccctt tgctctctgg ggtagccttc 240 agatcttggt gttttgaatt cttactatag atgtttttaa agttccaaag tcattgagtt 300 tcaatgttac ataaactcca ttacccgcat gttggggctt gatctcctgg ttattatctg 360 tgcttgagga aacacccaca gcagtctcta cccagaacag tttcctaaag aggcataccc 420 tcttcctcca ctggaaaata gtgcgttccc ttcctaccct gcacacccat cgcccccaca 480 ttgatggttt tcaaacagca aacttttcag ctaaagcttc aaaccatgat tggaatcagc 540 ctgtgttgga tttgtgattc agggtcatgg tgaccctgat ccagtttggg tggaaatcct 600 tcctaagtat cataagaagc atcttggcag agatgctttg gtggcagcca tgagctttgc 660 tggaggnctt gcttcccata gccttggctg tgggggcagg aactctgcca agcgaagggg 720 atgctgccct ggatcacaga agcctggtgg gggttgctcg tgttagagtg tcctggcttt 780 ctactgacca ctcttctcgg tgatagcctc tcttccctga attgtgactt atggaatgac 840 agtgcaggta ccaccgaggc tagcacaagt caaccctcca gctaaactgg atccctgaag 900 cctggtatca tgccaaaaaa ggctatggcg gctggcctcc ggaccatgct aaggccccct 960 ttggttgggg ggggtcaacc ttacccaccc aaaagggg 998 14 0 DNA Homo sapiens 14 000 15 20 DNA Artificial Antisense Oligonucleotide 15 acacaactgc tgcacccgcg 20 16 20 DNA Artificial Antisense Oligonucleotide 16 gatctgaagg ctatggattt 20 17 20 DNA Artificial Antisense Oligonucleotide 17 gtcaggctaa gttcatttct 20 18 20 DNA Artificial Antisense Oligonucleotide 18 tgggaaggaa aatcaccacc 20 19 20 DNA Artificial Antisense Oligonucleotide 19 ggcgacctac ctccagaaac 20 20 20 DNA Artificial Antisense Oligonucleotide 20 tgccaccttc cacaacactg 20 21 20 DNA Artificial Antisense Oligonucleotide 21 gataacttac cctccaagcc 20 22 20 DNA Artificial Antisense Oligonucleotide 22 attcatcagc tcatctatcc 20 23 20 DNA Artificial Antisense Oligonucleotide 23 gggacacaac tgctgcaccc 20 24 20 DNA Artificial Antisense Oligonucleotide 24 gcacagctct gccatggcgc 20 25 20 DNA Artificial Antisense Oligonucleotide 25 acgacagctc ctcggccagg 20 26 20 DNA Artificial Antisense Oligonucleotide 26 gaccggctcc ttgaagggct 20 27 20 DNA Artificial Antisense Oligonucleotide 27 cagaagttgt ggccgcacgg 20 28 20 DNA Artificial Antisense Oligonucleotide 28 tgcccacgtc tcattcaggc 20 29 20 DNA Artificial Antisense Oligonucleotide 29 gaactgctcc accacgttgc 20 30 20 DNA Artificial Antisense Oligonucleotide 30 tgcaggaact gctccaccac 20 31 20 DNA Artificial Antisense Oligonucleotide 31 cggcctgcag gaactgctcc 20 32 20 DNA Artificial Antisense Oligonucleotide 32 gtggtcgcag gccacctggg 20 33 20 DNA Artificial Antisense Oligonucleotide 33 gcacaccaag cacgtcttca 20 34 20 DNA Artificial Antisense Oligonucleotide 34 tgacagaagg aggccatgca 20 35 20 DNA Artificial Antisense Oligonucleotide 35 ctgcagcggg tggtcctgga 20 36 20 DNA Artificial Antisense Oligonucleotide 36 ggcagatgtg gcagatgcac 20 37 20 DNA Artificial Antisense Oligonucleotide 37 agcaggtctt atgctccacc 20 38 20 DNA Artificial Antisense Oligonucleotide 38 gggagagcag gtcttatgct 20 39 20 DNA Artificial Antisense Oligonucleotide 39 gtacatgaca gttagtttgt 20 40 20 DNA Artificial Antisense Oligonucleotide 40 ttgcagtcat ccgcacatcc 20 41 20 DNA Artificial Antisense Oligonucleotide 41 cctttctgtt tgcagtcatc 20 42 20 DNA Artificial Antisense Oligonucleotide 42 gtccaagaga gccttcattt 20 43 20 DNA Artificial Antisense Oligonucleotide 43 gtcgaggtgg tctctgaggc 20 44 20 DNA Artificial Antisense Oligonucleotide 44 ctgttgaccc tcttctcctc 20 45 20 DNA Artificial Antisense Oligonucleotide 45 gaatctgata aatggtgtca 20 46 20 DNA Artificial Antisense Oligonucleotide 46 tcactcttct tcttgaggag 20 47 20 DNA Artificial Antisense Oligonucleotide 47 tcaaggtctg gatctcactc 20 48 20 DNA Artificial Antisense Oligonucleotide 48 ctcttggtca ggctctgttc 20 49 20 DNA Artificial Antisense Oligonucleotide 49 ttgatgcttt ctccagaaac 20 50 20 DNA Artificial Antisense Oligonucleotide 50 ttgagattcc tcgcagtttt 20 51 20 DNA Artificial Antisense Oligonucleotide 51 ctggtggatg ccttttatca 20 52 20 DNA Artificial Antisense Oligonucleotide 52 gtgctctggt ggatgccttt 20 53 20 DNA Artificial Antisense Oligonucleotide 53 ggtctatggt gctctggtgg 20 54 20 DNA Artificial Antisense Oligonucleotide 54 acgctgggtc atgctctcca 20 55 20 DNA Artificial Antisense Oligonucleotide 55 tcacagggcg tgtggatttg 20 56 20 DNA Artificial Antisense Oligonucleotide 56 ttgttttaaa tccactaact 20 57 20 DNA Artificial Antisense Oligonucleotide 57 accttggcct tgagagatgt 20 58 20 DNA Artificial Antisense Oligonucleotide 58 tccaggagct caggtctgga 20 59 20 DNA Artificial Antisense Oligonucleotide 59 ttaatgtaat actccaggag 20 60 20 DNA Artificial Antisense Oligonucleotide 60 cagccacaga agctactgta 20 61 20 DNA Artificial Antisense Oligonucleotide 61 ttctgaggca tctcagccac 20 62 20 DNA Artificial Antisense Oligonucleotide 62 ggatgcggcc ggtagttctg 20 63 20 DNA Artificial Antisense Oligonucleotide 63 agagcagtat gtgaacctct 20 64 20 DNA Artificial Antisense Oligonucleotide 64 acctgagagc agtatgtgaa 20 65 20 DNA Artificial Antisense Oligonucleotide 65 tccacctccc agtagtggat 20 66 20 DNA Artificial Antisense Oligonucleotide 66 gcagctccac ctcccagtag 20 67 20 DNA Artificial Antisense Oligonucleotide 67 ttcttctgca gctccacctc 20 68 20 DNA Artificial Antisense Oligonucleotide 68 agttgttctt ctgcagctcc 20 69 20 DNA Artificial Antisense Oligonucleotide 69 acagaagttg ttcttctgca 20 70 20 DNA Artificial Antisense Oligonucleotide 70 cagatgccta ccccacagaa 20 71 20 DNA Artificial Antisense Oligonucleotide 71 gcggccgagc ctgctttctg 20 72 20 DNA Artificial Antisense Oligonucleotide 72 ttggtgttga accactccac 20 73 20 DNA Artificial Antisense Oligonucleotide 73 cagggttttc tccacgttat 20 74 20 DNA Artificial Antisense Oligonucleotide 74 aagccgtggt cacagttgag 20 75 20 DNA Artificial Antisense Oligonucleotide 75 aagaagatga caaagccgtg 20 76 20 DNA Artificial Antisense Oligonucleotide 76 aagtccaccc tgaacttata 20 77 20 DNA Artificial Antisense Oligonucleotide 77 atacccagaa agccgggtac 20 78 20 DNA Artificial Antisense Oligonucleotide 78 agtgtggcac cagcagaaaa 20 79 20 DNA Artificial Antisense Oligonucleotide 79 atggagagtg tggcaccagc 20 80 20 DNA Artificial Antisense Oligonucleotide 80 ctacagcctg cctacttggg 20 81 20 DNA Artificial Antisense Oligonucleotide 81 tccaaggaga gttctgcctg 20 82 20 DNA Artificial Antisense Oligonucleotide 82 tcaccccttt cctggctaaa 20 83 20 DNA Artificial Antisense Oligonucleotide 83 tatggatttt ctctaagagg 20 84 20 DNA Artificial Antisense Oligonucleotide 84 ttcagatcca agtggcccaa 20 85 20 DNA Artificial Antisense Oligonucleotide 85 ctgaaggcta ccccagagag 20 86 20 DNA Artificial Antisense Oligonucleotide 86 aagatctgaa ggctacccca 20 87 20 DNA Artificial Antisense Oligonucleotide 87 cgggtaatgg agtttatgta 20 88 20 DNA Artificial Antisense Oligonucleotide 88 tgctgtgggt gtttcctcaa 20 89 20 DNA Artificial Antisense Oligonucleotide 89 ctgaatcaca aatccaacac 20 90 20 DNA Artificial Antisense Oligonucleotide 90 accaggcttc tgtgatccag 20 91 20 DNA Artificial Antisense Oligonucleotide 91 cccttccaca ctgattaaga 20 92 20 DNA Artificial Antisense Oligonucleotide 92 cgggatggcc tctactccct 20 93 20 DNA Homo sapiens 93 cgcgggtgca gcagttgtgt 20 94 20 DNA Homo sapiens 94 agaaatgaac ttagcctgac 20 95 20 DNA Homo sapiens 95 ggtggtgatt ttccttccca 20 96 20 DNA Homo sapiens 96 cagtgttgtg gaaggtggca 20 97 20 DNA Homo sapiens 97 ggcttggagg gtaagttatc 20 98 20 DNA Homo sapiens 98 ggatagatga gctgatgaat 20 99 20 DNA Homo sapiens 99 gggtgcagca gttgtgtccc 20 100 20 DNA Homo sapiens 100 gcgccatggc agagctgtgc 20 101 20 DNA Homo sapiens 101 cctggccgag gagctgtcgt 20 102 20 DNA Homo sapiens 102 agcccttcaa ggagccggtc 20 103 20 DNA Homo sapiens 103 ccgtgcggcc acaacttctg 20 104 20 DNA Homo sapiens 104 gcctgaatga gacgtgggca 20 105 20 DNA Homo sapiens 105 gcaacgtggt ggagcagttc 20 106 20 DNA Homo sapiens 106 gtggtggagc agttcctgca 20 107 20 DNA Homo sapiens 107 ggagcagttc ctgcaggccg 20 108 20 DNA Homo sapiens 108 cccaggtggc ctgcgaccac 20 109 20 DNA Homo sapiens 109 tgaagacgtg cttggtgtgc 20 110 20 DNA Homo sapiens 110 tgcatggcct ccttctgtca 20 111 20 DNA Homo sapiens 111 tccaggacca cccgctgcag 20 112 20 DNA Homo sapiens 112 gtgcatctgc cacatctgcc 20 113 20 DNA Homo sapiens 113 ggtggagcat aagacctgct 20 114 20 DNA Homo sapiens 114 agcataagac ctgctctccc 20 115 20 DNA Homo sapiens 115 acaaactaac tgtcatgtac 20 116 20 DNA Homo sapiens 116 ggatgtgcgg atgactgcaa 20 117 20 DNA Homo sapiens 117 gatgactgca aacagaaagg 20 118 20 DNA Homo sapiens 118 aaatgaaggc tctcttggac 20 119 20 DNA Homo sapiens 119 gcctcagaga ccacctcgac 20 120 20 DNA Homo sapiens 120 gaggagaaga gggtcaacag 20 121 20 DNA Homo sapiens 121 tgacaccatt tatcagattc 20 122 20 DNA Homo sapiens 122 ctcctcaaga agaagagtga 20 123 20 DNA Homo sapiens 123 gagtgagatc cagaccttga 20 124 20 DNA Homo sapiens 124 gaacagagcc tgaccaagag 20 125 20 DNA Homo sapiens 125 gtttctggag aaagcatcaa 20 126 20 DNA Homo sapiens 126 aaaactgcga ggaatctcaa 20 127 20 DNA Homo sapiens 127 tgataaaagg catccaccag 20 128 20 DNA Homo sapiens 128 aaaggcatcc accagagcac 20 129 20 DNA Homo sapiens 129 ccaccagagc accatagacc 20 130 20 DNA Homo sapiens 130 tggagagcat gacccagcgt 20 131 20 DNA Homo sapiens 131 caaatccaca cgccctgtga 20 132 20 DNA Homo sapiens 132 agttagtgga tttaaaacaa 20 133 20 DNA Homo sapiens 133 acatctctca aggccaaggt 20 134 20 DNA Homo sapiens 134 tccagacctg agctcctgga 20 135 20 DNA Homo sapiens 135 tacagtagct tctgtggctg 20 136 20 DNA Homo sapiens 136 gtggctgaga tgcctcagaa 20 137 20 DNA Homo sapiens 137 cagaactacc ggccgcatcc 20 138 20 DNA Homo sapiens 138 agaggttcac atactgctct 20 139 20 DNA Homo sapiens 139 ttcacatact gctctcaggt 20 140 20 DNA Homo sapiens 140 atccactact gggaggtgga 20 141 20 DNA Homo sapiens 141 ctactgggag gtggagctgc 20 142 20 DNA Homo sapiens 142 gaggtggagc tgcagaagaa 20 143 20 DNA Homo sapiens 143 ggagctgcag aagaacaact 20 144 20 DNA Homo sapiens 144 tgcagaagaa caacttctgt 20 145 20 DNA Homo sapiens 145 ttctgtgggg taggcatctg 20 146 20 DNA Homo sapiens 146 cagaaagcag gctcggccgc 20 147 20 DNA Homo sapiens 147 gtggagtggt tcaacaccaa 20 148 20 DNA Homo sapiens 148 ataacgtgga gaaaaccctg 20 149 20 DNA Homo sapiens 149 ctcaactgtg accacggctt 20 150 20 DNA Homo sapiens 150 cacggctttg tcatcttctt 20 151 20 DNA Homo sapiens 151 tataagttca gggtggactt 20 152 20 DNA Homo sapiens 152 gtacccggct ttctgggtat 20 153 20 DNA Homo sapiens 153 ttttctgctg gtgccacact 20 154 20 DNA Homo sapiens 154 gctggtgcca cactctccat 20 155 20 DNA Homo sapiens 155 cccaagtagg caggctgtag 20 156 20 DNA Homo sapiens 156 caggcagaac tctccttgga 20 157 20 DNA Homo sapiens 157 tttagccagg aaaggggtga 20 158 20 DNA Homo sapiens 158 cctcttagag aaaatccata 20 159 20 DNA Homo sapiens 159 ttgggccact tggatctgaa 20 160 20 DNA Homo sapiens 160 ctctctgggg tagccttcag 20 161 20 DNA Homo sapiens 161 tggggtagcc ttcagatctt 20 162 20 DNA Homo sapiens 162 tacataaact ccattacccg 20 163 20 DNA Homo sapiens 163 ttgaggaaac acccacagca 20 164 20 DNA Homo sapiens 164 gtgttggatt tgtgattcag 20 165 20 DNA Homo sapiens 165 ctggatcaca gaagcctggt 20 166 20 DNA Homo sapiens 166 tcttaatcag tgtggaaggg 20 167 2304 DNA Homo sapiens CDS (40)..(1932) 167 cgcgggtgca gcagttgtgt cccgacccct gggagcgcc atg gca gag ctg tgc 54 Met Ala Glu Leu Cys 1 5 ccc ctg gcc gag gag ctg tcg tgc tcc atc tgc ctg gag ccc ttc aag 102 Pro Leu Ala Glu Glu Leu Ser Cys Ser Ile Cys Leu Glu Pro Phe Lys 10 15 20 gag ccg gtc acc act ccg tgc ggc cac aac ttc tgc ggg tcg tgc ctg 150 Glu Pro Val Thr Thr Pro Cys Gly His Asn Phe Cys Gly Ser Cys Leu 25 30 35 aat gag acg tgg gca gtc cag ggc tcg cca tac ctg tgc ccg cag tgc 198 Asn Glu Thr Trp Ala Val Gln Gly Ser Pro Tyr Leu Cys Pro Gln Cys 40 45 50 cgc gcc gtc tac cag gcg cga ccg cag ctg cac aag aac acg gtg ctg 246 Arg Ala Val Tyr Gln Ala Arg Pro Gln Leu His Lys Asn Thr Val Leu 55 60 65 tgc aac gtg gtg gag cag ttc ctg cag gcc gac ctg gcc cgg gag cca 294 Cys Asn Val Val Glu Gln Phe Leu Gln Ala Asp Leu Ala Arg Glu Pro 70 75 80 85 ccc gcc gac gtc tgg acg ccg ccc gcc cgc gcc tct gca ccc agc ccg 342 Pro Ala Asp Val Trp Thr Pro Pro Ala Arg Ala Ser Ala Pro Ser Pro 90 95 100 aat gcc cag gtg gcc tgc gac cac tgc ctg aag gag gcc gcc gtg aag 390 Asn Ala Gln Val Ala Cys Asp His Cys Leu Lys Glu Ala Ala Val Lys 105 110 115 acg tgc ttg gtg tgc atg gcc tcc ttc tgt cag gag cac ctg cag ccg 438 Thr Cys Leu Val Cys Met Ala Ser Phe Cys Gln Glu His Leu Gln Pro 120 125 130 cac ttc gac agc ccc gcc ttc cag gac cac ccg ctg cag ccg ccc gtt 486 His Phe Asp Ser Pro Ala Phe Gln Asp His Pro Leu Gln Pro Pro Val 135 140 145 cgc gac ctg ttg cgc cgc aaa tgt tcc cag cac aat cgg ctg cgg gaa 534 Arg Asp Leu Leu Arg Arg Lys Cys Ser Gln His Asn Arg Leu Arg Glu 150 155 160 165 ttt ttc tgc ccc gag cac agc gag tgc atc tgc cac atc tgc ctg gtg 582 Phe Phe Cys Pro Glu His Ser Glu Cys Ile Cys His Ile Cys Leu Val 170 175 180 gag cat aag acc tgc tct ccc gcg tcc ctg agc cag gcc agc gcc gac 630 Glu His Lys Thr Cys Ser Pro Ala Ser Leu Ser Gln Ala Ser Ala Asp 185 190 195 ctg gag gcc acc ctg agg cac aaa cta act gtc atg tac agt cag atc 678 Leu Glu Ala Thr Leu Arg His Lys Leu Thr Val Met Tyr Ser Gln Ile 200 205 210 aac ggg gcg tcg aga gca ctg gat gat gtg aga aac agg cag cag gat 726 Asn Gly Ala Ser Arg Ala Leu Asp Asp Val Arg Asn Arg Gln Gln Asp 215 220 225 gtg cgg atg act gca aac aga aag gtg gag cag cta caa caa gaa tac 774 Val Arg Met Thr Ala Asn Arg Lys Val Glu Gln Leu Gln Gln Glu Tyr 230 235 240 245 acg gaa atg aag gct ctc ttg gac gcc tca gag acc acc tcg aca agg 822 Thr Glu Met Lys Ala Leu Leu Asp Ala Ser Glu Thr Thr Ser Thr Arg 250 255 260 aag ata aag gaa gag gag aag agg gtc aac agc aag ttt gac acc att 870 Lys Ile Lys Glu Glu Glu Lys Arg Val Asn Ser Lys Phe Asp Thr Ile 265 270 275 tat cag att ctc ctc aag aag aag agt gag atc cag acc ttg aag gag 918 Tyr Gln Ile Leu Leu Lys Lys Lys Ser Glu Ile Gln Thr Leu Lys Glu 280 285 290 gag att gaa cag agc ctg acc aag agg gat gag ttc gag ttt ctg gag 966 Glu Ile Glu Gln Ser Leu Thr Lys Arg Asp Glu Phe Glu Phe Leu Glu 295 300 305 aaa gca tca aaa ctg cga gga atc tca aca aag cca gtc tac atc ccc 1014 Lys Ala Ser Lys Leu Arg Gly Ile Ser Thr Lys Pro Val Tyr Ile Pro 310 315 320 325 gag gtg gaa ctg aac cac aag ctg ata aaa ggc atc cac cag agc acc 1062 Glu Val Glu Leu Asn His Lys Leu Ile Lys Gly Ile His Gln Ser Thr 330 335 340 ata gac ctc aaa aac gag ctg aag cag tgc atc ggg cgg ctc cag gag 1110 Ile Asp Leu Lys Asn Glu Leu Lys Gln Cys Ile Gly Arg Leu Gln Glu 345 350 355 ctc acc ccc agt tca ggt gac cct gga gag cat gac cca gcg tcc aca 1158 Leu Thr Pro Ser Ser Gly Asp Pro Gly Glu His Asp Pro Ala Ser Thr 360 365 370 cac aaa tcc aca cgc cct gtg aag aag gtc tcc aaa gag gaa aag aaa 1206 His Lys Ser Thr Arg Pro Val Lys Lys Val Ser Lys Glu Glu Lys Lys 375 380 385 tcc aag aaa cct ccc cct gtc cct gcc tta ccc agc aag ctt ccc acg 1254 Ser Lys Lys Pro Pro Pro Val Pro Ala Leu Pro Ser Lys Leu Pro Thr 390 395 400 405 ttt gga gcc ccg gaa cag tta gtg gat tta aaa caa gct ggc ttg gag 1302 Phe Gly Ala Pro Glu Gln Leu Val Asp Leu Lys Gln Ala Gly Leu Glu 410 415 420 gct gca gcc aaa gcc acc agc tca cat ccg aac tca aca tct ctc aag 1350 Ala Ala Ala Lys Ala Thr Ser Ser His Pro Asn Ser Thr Ser Leu Lys 425 430 435 gcc aag gtg ctg gag acc ttc ctg gcc aag tcc aga cct gag ctc ctg 1398 Ala Lys Val Leu Glu Thr Phe Leu Ala Lys Ser Arg Pro Glu Leu Leu 440 445 450 gag tat tac att aaa gtc atc ctg gac tac aac acc gcc cac aac aaa 1446 Glu Tyr Tyr Ile Lys Val Ile Leu Asp Tyr Asn Thr Ala His Asn Lys 455 460 465 gtg gct ctg tca gag tgc tat aca gta gct tct gtg gct gag atg cct 1494 Val Ala Leu Ser Glu Cys Tyr Thr Val Ala Ser Val Ala Glu Met Pro 470 475 480 485 cag aac tac cgg ccg cat ccc cag agg ttc aca tac tgc tct cag gtg 1542 Gln Asn Tyr Arg Pro His Pro Gln Arg Phe Thr Tyr Cys Ser Gln Val 490 495 500 ctg ggc ctg cac tgc tac aag aag ggg atc cac tac tgg gag gtg gag 1590 Leu Gly Leu His Cys Tyr Lys Lys Gly Ile His Tyr Trp Glu Val Glu 505 510 515 ctg cag aag aac aac ttc tgt ggg gta ggc atc tgc tac gga agc atg 1638 Leu Gln Lys Asn Asn Phe Cys Gly Val Gly Ile Cys Tyr Gly Ser Met 520 525 530 aac cgg cag ggc cca gaa agc agg ctc ggc cgc aac agc gcc tcc tgg 1686 Asn Arg Gln Gly Pro Glu Ser Arg Leu Gly Arg Asn Ser Ala Ser Trp 535 540 545 tgc gtg gag tgg ttc aac acc aag atc tct gcc tgg cac aat aac gtg 1734 Cys Val Glu Trp Phe Asn Thr Lys Ile Ser Ala Trp His Asn Asn Val 550 555 560 565 gag aaa acc ctg ccc tcc acc aag gcc acg cgg gtg ggc gtg ctt ctc 1782 Glu Lys Thr Leu Pro Ser Thr Lys Ala Thr Arg Val Gly Val Leu Leu 570 575 580 aac tgt gac cac ggc ttt gtc atc ttc ttc gct gtt gcc gac aag gtc 1830 Asn Cys Asp His Gly Phe Val Ile Phe Phe Ala Val Ala Asp Lys Val 585 590 595 cac ctg atg tat aag ttc agg gtg gac ttt act gag gct ttg tac ccg 1878 His Leu Met Tyr Lys Phe Arg Val Asp Phe Thr Glu Ala Leu Tyr Pro 600 605 610 gct ttc tgg gta ttt tct gct ggt gcc aca ctc tcc atc tgc tcc ccc 1926 Ala Phe Trp Val Phe Ser Ala Gly Ala Thr Leu Ser Ile Cys Ser Pro 615 620 625 aag tag gcaggctgta ggcacttggg ctgactgcct gcagaagtcc caagacccta 1982 Lys 630 gtgaaaatac agcaggcaga actctccttg gataattccc ccaagaggtc cccaaggatt 2042 gggagcatgg gaggggagct ggcgggaggg tgggaggtgg gatttagcca ggaaaggggt 2102 gagagtgatt gtgttgtggg cgaggaggcg tttccacccc ctggtgccta tcagggcagg 2162 gtgacctact ccccattgtt ctggaaatct ccaggctgct gggcagctgg gcagctgggc 2222 agagctctgg gaagtgaagt catgagtgcc cgattcctct tagagaaaat ccatagcctt 2282 cagatcttgg tgttttgaat tc 2304 168 630 PRT Homo sapiens 168 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding estrogen-responsive finger protein, wherein said compound specifically hybridizes with said nucleic acid molecule encoding estrogen-responsive finger protein (SEQ ID NO: 4) and inhibits the expression of estrogen-responsive finger protein.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding estrogen-responsive finger protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of estrogen-responsive finger protein.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding estrogen-responsive finger protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of estrogen-responsive finger protein.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding estrogen-responsive finger protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of estrogen-responsive finger protein.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding estrogen-responsive finger protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of estrogen-responsive finger protein.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of estrogen-responsive finger protein in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of estrogen-responsive finger protein is inhibited.
 19. A method of screening for a modulator of estrogen-responsive finger protein, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding estrogen-responsive finger protein with one or more candidate modulators of estrogen-responsive finger protein, and b. identifying one or more modulators of estrogen-responsive finger protein expression which modulate the expression of estrogen-responsive finger protein.
 20. The method of claim 19 wherein the modulator of estrogen-responsive finger protein expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of estrogen-responsive finger protein in a sample using at least one of the primers comprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with estrogen-responsive finger protein comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of estrogen-responsive finger protein is inhibited.
 24. The method of claim 23 wherein the disease or condition is a hyperproliferative disorder. 